The present invention, in some embodiments thereof, relates to Gemini-virus constructs capable of symptomless, systemic spread in plant hosts.
Plants may be genetically engineered for a variety of purposes including for the generation of plants with enhanced viral resistance, for the development of abiotic stress tolerant plants, for commercially improved plants and for the expression of heterologous polypeptides for pharmaceutical and industrial purposes.
Present techniques for DNA delivery into plants include direct as well as indirect methods. However, each of these delivery methods is not without limitations. The direct DNA delivery systems such as particle bombardment, silicon carbide whisker technology and electroporation tend to result in integration of multiple copies of transgenes and are considered to be limited, unpredictable and transient. Indirect approaches such as Agrobacterium oftentimes result in integration of multiple copies of the foreign DNA into the plant genome along with unwanted sequences from the vector ‘backbone’.
Integration of foreign DNA into the plant genome to become a heritable trait raises many risks. Traits beneficial to crops may, through horizontal gene transfer or hybridization through breeding with wild relatives, provide wild plants with unwanted competitive advantages. Also, Transformation with Agrobacterium is a complex process which requires elimination of false positives arising from the growth of Agrobacterium in host tissues, and selection of transformed plants. The use of antibiotic resistance as a marker in the development of transgenic crops has also raised concerns regarding the increase of antibiotic resistance in the environment through horizontal transfer of antibiotic resistance genes to soil micro-organisms. Scientists now have the means to remove marker genes before a crop plant is developed for commercial use, but these means involve further costs and tedious procedures. In addition, several species or varieties of plants are still difficult to transform.
Infection of plants with modified viruses is simpler and quicker than the regeneration of stably transformed plants, since plant viruses are small and easy to manipulate, have the inherent ability to enter the plant cell, and will multiply to produce a high copy number of the gene of interest. Viral vectors have been engineered for delivery of genetic material and expression of recombinant proteins in plants. Viral expression systems are considered transient expression systems since the viral vectors are not integrated into the genome of the host. However, viral vectors still hold many limitations. Plant viral vectors have the potential to cause disease in their plant hosts, they posses the ability to naturally spread between plants in the field, and in some cases, can be spread through pollen or seed to the next generation. Viral vectors are also limited in their systemic spread in the plant, in host ranges, expression stability, and in the size of insert which can be tolerated. Finally, like transgenic plants, modified viruses are classified as a Genetically Modified Organism (GMO) and thus are subject to regulatory and moral constraints.
Viruses belonging to the family Geminiviridae carry one or two circular single-stranded (ss) DNA genomes and are insect-transmissible. Begomoviruses, Curtoviruses, Mastreviruses and Topocuviruses are genera within this family which have been classified according to genome organization, insect transmissibility and host range. Begomoviruses can carry a monopartite or bipartite genome and are whitefly-transmissible. The association of satellite DNAs with monopartite begomoviruses is addressed further on.
Tomato yellow leaf curl virus (TYLCV) is a monopartite begomovirus. It carries six overlapping open reading frames (ORFs) transcribed bi-directionally from an intergenic region (IR; also termed “common region”), serving as the viral origin of replication and as a bi-directional promoter. The IR is about 300-bp long and carries the universal motif TAATATT/AC and a binding site for the replicase-associated protein (REP). Two ORFs are expressed in viral orientation (V1, V2) and four ORFs in the complementary orientation (C1 to C4). Each gene product appears to participate in more than one function. The viral ssDNA is transmitted to the plant and converted into double-stranded (ds) DNA (replicative form) by the host machinery. The dsDNA replicates and expresses viral mRNAs and proteins. Viral genes are not involved in this stage of replication (dsDNA to dsDNA). However, when rolling circle replication (RCR) is initiated by REP, RCR intermediates may serve as templates for dsDNA replication, dependent on host gene activity. At the next stage of replication, the numerous viral dsDNAs serve as templates for RCR, producing progeny ssDNA. ORF C1 (also termed rep) is essential for the initiation of RCR. The progeny ssDNA molecules are then encapsidated, transported to other tissues or transmitted to other plants. At least three viral gene products are implicated in viral movement within the plant: V2, the coat protein (CP) V1, and C4. The viral genes V1, V2, C4 and probably C2 are implicated in symptom appearance and disease severity. In summary, five of the viral ORFs have no direct role in viral DNA replication or expression (some have auxiliary roles), whereas the sixth ORF (rep) is involved only in the rolling-circle phase. Movement and pathogenicity, however, require the activity of viral gene products.
The association of viral satellites with RNA viruses is a well-documented phenomenon. Satellites, either encapsidated or in the form of naked nucleic acid, depend on a helper virus for their replication. A geminivirus-associated, 682-base-long DNA satellite was first reported by Dry et al. in 1997, Proc. Natl. Acad. Sci. USA 94, 7088-7093. This satellite was associated with an Australian type of TYLCV and did not share sequence homology with the helper virus. However, the satellite carried two motifs that also reside in the IR of the helper virus: the universal stem-loop motif and the REP-binding motif. Other gemini-associated satellites have been discovered since then (Briddon et al., 2001, Virology 285, 234-243; Mansoor et al., 1999, Virology 259, 190-199; Zhou et al., 2003, J. Gen. Virol. 84, 237-247. All of these satellite DNAs (generally referred to as DNAβ and DNA-1) are about half the size of the viral DNA, and carry, in addition to the aforementioned motifs, an ORF termed βC1. The role of the putative βC1 protein has not yet been conclusively determined but it is probably involved in movement, nuclear localization and silencing suppression (Cui et al., 2005, J. Virol. 79, 10764-10775). In many cases of geminiviral infection, the satellite determines symptom severity. Geminiviral satellites are encapsidated and replicated via factors provided by helper viruses. A satellite associated with a particular virus may be supported for replication by other geminiviruses as well.
International Patent Application WO2007/141790 teaches Gemini-virus based constructs wherein the inserted sequence to be expressed is flanked by a non-contiguous nucleic acid sequence encoding a Geminivirus replicase.